Novel motor proteins and methods for their use

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of HsKifC2, antibodies to HsKifC2, methods of screening for HsKifC2 modulators using biologically active HsKifC2, and kits for screening for HsKifC2 modulators

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/721,137, filed Nov. 22, 2000, the fulldisclosures of which are incorporated herein by reference in theirentirety for all purposes.

FIELD OF THE INVENTION

[0002] The invention provides isolated nucleic acid and amino acidsequences of HsKifC2, methods of detecting HsKifC2 and screening forHsKifC2 modulators using biologically active HsKifC2, and kits forscreening for HsKifC2 modulators.

BACKGROUND OF THE INVENTION

[0003] The kinesin superfamily is an extended family of relatedmicrotubule motor proteins. It can be classified into at least 8subfamilies based on primary amino acid sequence, domain structure,velocity of movement, and cellular function. This family is exemplifiedby “true” kinesin, which was first isolated from the axoplasm of squid,where it is believed to play a role in anterograde axonal transport ofvesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet.27:319-351 (1993)). Kinesin uses ATP to generate force and directionalmovement associated with microtubules.

[0004] Within this functional group of kinesins resides a group ofkinesins from several organisms that share significant sequencehomology, the Kin I subfamily, and that function to destabilizemicrotubule ends. These include H. sapiens MCAK (also known as mitoticcentromere-associated kinesin or HsMCAK), X. laevis MCAK, and C. griseusMCAK.

[0005] During anaphase A, disjoined sister chromatids migrate poleward.This poleward movement is driven by kinetochores and is accompanied bythe depolymerization of microtubules attached to the migratingchromatids. The kinesin MCAK plays an important role in this motilityand may promote disassembly of microtubules attached to kinetochores ofmitotic chromosomes.

[0006] The HsMCAK gene has a predicted 723 amino acid open readingframe, encoding a 81 kDa protein that shares 79.2% homology with hamsterMCAK. HsMCAK is expressed in tissues containing dividing cells, such asthymus, testis, small intestine, colon (mucosal lining), and placentaGenes for the Xenopus and hamster homologs of MCAK has also been clonedand characterized.

[0007] Defects in function of these proteins may result in cell cyclearrest. As such, compounds that modulate the activity of these kinesinsmay affect cellular proliferation. The present invention provides anovel method to identify such compounds.

[0008] The discovery of a new kinesin motor protein, and moreparticularly, one in the MCAK subfamily, and the polynucleotidesencoding it satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention, and treatment of cancer,neurological disorders, and disorders of vesicular transport.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the discovery of a new humankinesin motor protein, HsKifC2, the polynucleotide encoding HsKifC2, andthe use of these compositions for the diagnosis, treatment, orprevention of cancer, neurological disorders, and disorders of vesiculartransport.

[0010] In one aspect, the invention provides an isolated nucleic acidsequence encoding a kinesin superfamily motor protein, wherein the motorprotein has the following properties: (i) the protein's activityincludes microtubule stimulated ATPase activity; and (ii) the proteinhas a sequence that has greater than 70% amino acid sequence identity toSEQ ID NO:2 or SEQ ID NO:4 as measured using a sequence comparisonalgorithm. In one embodiment, the protein further specifically binds topolyclonal antibodies raised against SEQ ID NO:2 or SEQ ID NO:4.

[0011] In one embodiment, the nucleic acid encodes HsKifC2 or a fragmentthereof In another embodiment, the nucleic acid encodes SEQ ID NO:2 orSEQ ID NO:4. In another embodiment, the nucleic acid has a nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3.

[0012] In one aspect, the nucleic acid comprises a sequence whichencodes an amino acid sequence which has greater than 70% sequenceidentity with SEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%,more preferably greater than 90%, more preferably greater than 95% or,in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2or SEQ ID NO:4.

[0013] In one embodiment, the nucleic acid comprises a sequence whichhas greater than 55 or 60% sequence identity with SEQ ID NO:1 or SEQ IDNO:3, preferably greater than 70%, more preferably greater than 80%,more preferably greater than 90 or 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:3. In anotherembodiment provided herein, the nucleic acid hybridizes under stringentconditions to a nucleic acid having a sequence or complementary sequenceof SEQ ID NO:1 or SEQ ID NO:3.

[0014] In another aspect, the invention provides an expression vectorcomprising a nucleic acid encoding a kinesin superfamily motor protein,wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm. The invention further provides a hostcell transfected with the vector.

[0015] In another aspect, the invention provides an isolated kinesinsuperfamily motor protein, wherein the protein has one or more of theproperties described above. In one embodiment, the protein specificallybinds to polyclonal antibodies generated against a motor domain, taildomain or other fragment of HsKifC2. In another embodiment, the proteincomprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0016] In one aspect, the protein provided herein comprises an aminoacid sequence which has greater than 70% sequence identity with SEQ IDNO:2 or SEQ ID NO:4, preferably greater than 80%, more preferablygreater than 90%, more preferably greater than 95% or, in anotherembodiment, has 98 to 100% sequence identity with SEQ ID NO:2 or SEQ IDNO:4.

[0017] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or afragment thereof and more particularly, the motor domain of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragment thereof.

[0018] In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

[0019] The phrase “use ADP or phosphate” means that the ADP or phosphateare directly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

[0020] Preferably, the target protein either directly or indirectlyproduces ADP or phosphate and comprises a motor domain. More preferably,the target protein comprises a kinesin superfamily motor protein asdescribed above and most preferably, the target protein comprisesHsKifC2 or a fragment thereof.

[0021] Also provided are modulators of the target protein includingagents for the treatment of cellular proliferation, including cancer,hyperplasias, restenosis, cardiac hypertrophy, immune disorders andinflammation. The agents and compositions provided herein can be used invariety of applications which include the formulation of sprays,powders, and other compositions. Also provided herein are methods oftreating cellular proliferation disorders such as cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation, fortreating disorders associated with HsKifC2 activity, and for inhibitingHsKifC2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows an embodiment of a nucleic acid sequence encodingHsKifC2 (SEQ ID NO:1).

[0023]FIG. 2 shows the predicted amino acid sequence of HsKifC2 (SEQ IDNO:2)

[0024]FIG. 3 shows an embodiment of a nucleic acid sequence encodingHsKifC2 motor domain fragment (SEQ ID NO:3).

[0025]FIG. 4 shows the predicted amino acid sequence of HsKifC2 motordomain fragment (SEQ ID NO:4).

[0026]FIG. 5 shows the qualitative tissue expression profile of HsKifC2in a variety of tissues. The results indicate that expression of KifC2is extremely specific and restricted to adult brain and colon. Weakexpression can also be detected in other tissues such as skin. Theprofile may be generated using end-point PCR together with cDNA dilutionto generate a gross evaluation of RNA expression across 24 humantissues. Plates containing the dilutions of cDNA can be obtained fromOrigene Technologies and techniques for producing such profiles areknown in the art. Alternatively, as will appreciated by those skilled inthe art, PCR with Taqman® real time fluorescence detection can be usedto produce the profiles.

DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Definitions

[0028] “ADP” refers to adenosine diphosphate and also includes ADPanalogs, including, but not limited to, deoxyadenosine diphosphate(dADP) and adenosine analogs. “Antibody” refers to a polypeptidesubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof which specifically bind and recognize an analyte(antigen). The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes. Light chainsare classified as either kappa or lambda Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Theterm antibody also includes antibody fragments either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies.

[0029] An “anti-HsKifC2” antibody is an antibody or antibody fragmentthat specifically binds a polypeptide encoded by the HsKifC2 gene, cDNA,or a subsequence thereof.

[0030] “Biologically active” target protein refers to a target proteinthat has one or more of kinesin protein's biological activities,including, but not limited to microtubule stimulated ATPase activity, astested, e.g., in an ATPase assay. Biological activity can also bedemonstrated in a microtubule gliding assay or a microtubule bindingassay. “ATPase activity” refers to ability to hydrolyze ATP. Otheractivities include polymerization/depolymerization (effects onmicrotubule dynamics), binding to other proteins of the spindle, bindingto proteins involved in cell-cycle control, or serving as a substrate toother enzymes, such as kinases or proteases and specific kinesincellular activities, such as chromosome congregation, axonal transport,etc.

[0031] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains a target protein or a fragment thereof ornucleic acid encoding a target protein or a fragment thereof. Biologicalsamples may also include sections of tissues such as frozen sectionstaken for histological purposes. A biological sample comprises at leastone cell, preferably plant or vertebrate. Embodiments include cellsobtained from a eukaryotic organism, preferably eukaryotes such asfungi, plants, insects, protozoa, birds, fish, reptiles, and preferablya mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and mostpreferably a primate such as chimpanzees or humans.

[0032] A “comparison window” includes reference to a segment of any oneof the number of contiguous positions selected from the group consistingof from 25 to 600, usually about 50 to about 200, more usually about 100to about 150 in which a sequence may be compared to a reference sequenceof the same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

[0033] This algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W in the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as the neighborhood word score threshold(Altschul et al, supra.). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

[0034] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0035] Another example of a useful algorithm implementation is PILEUP.PILEUP creates a multiple sequence alignment from a group of relatedsequences using progressive, pairwise alignments. It can also plot adendrogram showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). As a general rule, PileUp can alignup to 500sequences, with any single sequence in the final alignment restricted toa maximum length of 7,000 characters.

[0036] The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster can then be aligned to the next mostrelated sequence or cluster of aligned sequences. Two clusters ofsequences can be aligned by a simple extension of the pairwise alignmentof two individual sequences. A series of such pairwise alignments thatincludes increasingly dissimilar sequences and clusters of sequences ateach iteration produces the final alignment.

[0037] “Variant” applies to both amino acid and nucleic acid sequences.With respect to particular nucleic acid sequences, conservativelymodified variants refers to those nucleic acids which encode identicalor essentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

[0038] Also included within the definition of target proteins of thepresent invention are amino acid sequence variants of wild-type targetproteins. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding the target protein, using cassette or PCR mutagenesisor other techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell culture.Variant target protein fragments having up to about 100-150 amino acidresidues may be prepared by in vitro synthesis using establishedtechniques. Amino acid sequence variants are characterized by thepredetermined nature of the variation, a feature that sets them apartfrom naturally occurring allelic or interspecies variation of the targetprotein amino acid sequence. The variants typically exhibit the samequalitative biological activity as the naturally occurring analogue,although variants can also be selected which have modifiedcharacteristics.

[0039] Amino acid substitutions are typically of single residues;insertions usually will be on the order of from about 1 to about 20amino acids, although considerably longer insertions may be tolerated.Deletions range from about 1 to about 20 residues, although in somecases, deletions may be much longer.

[0040] Substitutions, deletions, and insertions or any combinationsthereof may be used to arrive at a final derivative. Generally, thesechanges are done on a few amino acids to minimize the alteration of themolecule. However, larger characteristics may be tolerated in certaincircumstances.

[0041] Individual substitutions, to a nucleic acid, peptide,polypeptide, or protein sequence which alters a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art (Henikoff and Henikoff (Proc. Natl.Acad. Sci. USA 89; 10915-10919 (1992))).

[0042] “Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Includes cytoskeletal polymers (e.g., actin filaments,microtubules, intermediate filaments, myosin fragments), molecularmotors (e.g., kinesins, myosins, dyneins), cytoskeleton associatedregulatory proteins (e.g., tropomysin, alpha-actinin) and cytoskeletalassociated binding proteins (e.g., microtubules associated proteins,actin binding proteins).

[0043] “Cytoskeletal function” refers to biological roles of thecytoskeleton, including but not limited to the providing of structuralorganization (e.g., microvilli, mitotic spindle) and the mediation ofmotile events within the cell (e.g., muscle contraction, mitoticchromosome movements, contractile ring formation and function,pseudopodal movement, active cell surface deformations, vesicleformation and translocation.)

[0044] A “diagnostic” as used herein is a compound, method, system, ordevice that assists in the identification and characterization of ahealth or disease state. The diagnostic can be used in standard assaysas is known in the art.

[0045] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0046] “High stringency conditions” may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agentsuch as formamide, for example, 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

[0047] “High throughput screening” as used herein refers to an assaywhich provides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

[0048] By “host cell” is meant a cell that contains an expression vectorand supports the replication or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells such as CHO, HeLaand the like, or plant cells. Both primary cells and cultured cell linesare included in this definition.

[0049] The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

[0050] The terms “identical” or percent “identity”, in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is atleast 50 amino acids in length. This definition also refers to thereverse complement of a test nucleic acid sequence, provided that thetest sequence has a designated or substantial identity to a referencesequence. Preferably, the percent identity exists over a region of thesequence that is at least about 25 nucleotides in length, morepreferably over a region that is at least 50 nucleotides in length.

[0051] When percentage of sequence identity is used in reference toproteins or peptides, it is recognized that residue positions that arenot identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties (e.g., charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Means for making this adjustment are well known tothose of skill in the art. The scoring of conservative substitutions canbe calculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988)

[0052] The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated gene, the nucleic acid of interest is separated from openreading frames which flank the gene of interest and encode proteinsother than the protein of interest. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

[0053] A “label” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include fluorescent proteins such as green,yellow, red or blue fluorescent proteins, radioisotopes such as ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins forwhich antisera or monoclonal antibodies are available (e.g., thepolypeptide of SEQ ID NO:2 can be made detectable, e.g., byincorporating a radio-label into the peptide, and used to detectantibodies specifically reactive with the peptide).

[0054] A “labeled nucleic acid probe or oligonucleotide” is one that isbound, either covalently, through a linker, or through ionic, van derWaals, or hydrogen bonds to a label such that the presence of the probemay be detected by detecting the presence of the label bound to theprobe.

[0055] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20μg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0056] “Modulators,” “inhibitors,” and “activators of a target protein”refer to modulatory molecules identified using in vitro and in vivoassays for target protein activity. Such assays include ATPase activity,microtubule gliding, microtubule depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

[0057] “Molecular motor” refers to a molecule that utilizes chemicalenergy to generate mechanical force. According to one embodiment, themolecular motor drives the motile properties of the cytoskeleton.

[0058] The phrase “motor domain” refers to the domain of a targetprotein that confers membership in the kinesin superfamily of motorproteins as determined by alignment with the motor domain of truekinesin.

[0059] The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

[0060] “Nucleic acid probe or oligonucleotide” is defined as a nucleicacid capable of binding to a target nucleic acid of complementarysequence through one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, T, or U) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

[0061] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A target protein comprises a polypeptide demonstrated to haveat least microtubule stimulated ATPase activity and, preferably thatalso binds to an antibody selectively immunoreactive with HsKifC2 orwhose sequence is derived from HsKifC2. by mutagenesis and/orrecombination. Amino acids may be referred to herein by either theircommonly known one or three letter symbols. Nucleotides, likewise, maybe referred to by their commonly accepted single-letter codes, i.e., theone-letter symbols recommended by the IUPAC-IUB.

[0062] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA box element. A promoter also optionally includes distal enhanceror repressor elements which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isunder environmental or developmental regulation. The term “operablylinked” refers to a functional linkage between a nucleic acid expressioncontrol sequence (such as a promoter, or array of transcription factorbinding sites) and a second nucleic acid sequence, wherein theexpression control sequence directs transcription of the nucleic acidcorresponding to the second sequence.

[0063] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding moieties typically have an affinity for one another ofat least 10⁶ M⁻¹. Preferred antibodies for use in diagnostics ortherapeutics often have high affinities such as 10⁷, 10⁸, 10⁹ or 10¹⁰M⁻¹. Specific binding to an antibody under such conditions may requirean antibody that is selected for its specificity for a particularprotein. For example, antibodies raised to HsKifC2 with the amino acidsequence encoded in SEQ ID NO:2 or SEQ ID NO:4 can be selected to obtainonly those antibodies that are specifically immunoreactive with HsKifC2and not with other proteins, except for polymorphic variants, orthologs,alleles, and closely related homologues of HsKifC2. This selection maybe achieved by subtracting out antibodies that cross react withmolecules, for example, such as C. elegans unc-104 and human Kif1A. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

[0064] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0065] “Test composition” (used interchangeably herein with “candidateagent” and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

[0066] A “therapeutic” as used herein refers to a compound which isbelieved to be capable of modulating the cytoskeletal system in vivowhich can have application in either human or animal disease. Modulationof the cytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disease such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

[0067] II. The Target Protein

[0068] The present invention provides for the first time a nucleic acidencoding human HsKifC2. This protein is a member of the kinesinsuperfamily of motor proteins and the C-terminal subfamily. Morespecifically, the HsKifC2 sequence of FIG. 2 or FIG. 4 sharesapproximately 50% identity with various kinesins, being closest insequence to mouse KifC2.

[0069] In one aspect, HsKifC2 can be defined by having at least one orpreferably more than one of the following functional and structuralcharacteristics. Functionally, HsKifC2 will have microtubule-stimulatedATPase activity, and microtubule motor activity that is ATP dependent.HsKifC2 activity can also be described in terms of its ability to bindmicrotubules.

[0070] The novel nucleotides sequences provided herein encode HsKifC2 orfragments thereof. Thus, in one aspect, the nucleic acids providedherein are defined by the novel proteins provided herein. The proteinprovided herein comprises an amino acid sequence which has one or moreof the following characteristics: greater than 70% sequence identitywith SEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%, morepreferably greater than 90%, more preferably greater than 95% or, inanother embodiment, has 98 to 100% sequence identity with SEQ ID NO:2 orSEQ ID NO:4. As described above, when describing the nucleotide in termsof SEQ ID NO:1 or SEQ ID NO:3, the sequence identity can be the samepercentages or slightly lower due to the degeneracy in the genetic code.The invention also includes fragments of the nucleotide sequence shownin FIG. 1 (SEQ ID NO:1) having at least 10, 15, 20, 25, 50, 100, 1000 or2000 contiguous nucleotides from SEQ ID NO:1 or a degenerate formthereof. Some fragments include the sequence encoding the motor domainwhich occurs approximately between positions 1207 and 2184 of SEQ IDNO:1 (determined by sequence comparison of the motor domain of otherkinesins). Some such fragments can be used as hybridization probes orprimers. Unless otherwise apparent from the context, reference tonucleotide sequences shown in the Figures or sequences can refer to thesequence shown, its perfect complement or a duplex of the two strands.Also included within the definition of the target proteins are aminoacid sequence variants of wild-type target proteins.

[0071] Portions of the HsKifC2 nucleotide sequence may be used toidentify polymorphic variants, orthologs, alleles, and homologues ofHsKifC2. This identification can be made in vitro, e.g., under stringenthybridization conditions and sequencing, or by using the sequenceinformation in a computer system for comparison with other nucleotidesequences. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below, with BLAST as apreferred algorithm.

[0072] As will be appreciated by those in the art, the target proteinscan be made in a variety of ways, including both synthesis de novo andby expressing a nucleic acid encoding the protein.

[0073] Target proteins of the present invention may also be modified ina way to form chimeric molecules comprising a fusion of a target proteinwith a tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al. (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) Bio Technology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.

[0074] The biological activity of any of the peptides provided hereincan be routinely confirmed by the assays provided herein such as thosewhich assay ATPase activity or microtubule binding activity. In oneembodiment, polymorphic variants, alleles, and orthologs, homologues ofHsKifC2 are confirmed by using a ATPase or microtubule binding assays asknown in the art.

[0075] The isolation of biologically active HsKifC2 for the first timeprovides a means for assaying for modulators of this kinesin superfamilyprotein. Biologically active HsKifC2 is useful for identifyingmodulators of HsKifC2 or fragments thereof and kinesin superfamilymembers using in vitro assays such as microtubule gliding assays, ATPaseassays (Kodama et al., J. Biochem. 99:1465-1472 (1986); Stewart et al.,Proc. Nat'l Acad. Sci. USA 90:5209-5213 (1993)), and binding assaysincluding microtubule binding assays (Vale et al., Cell 42:39-50(1985)). In vivo assays and uses are provided herein as well. Alsoprovided herein are methods of identifying candidate agents which bindto HsKifC2 and portions thereof.

[0076] Some portions or fragments of HsKifC2 include at least 7, 10, 15,20, 35, 50, 100, 250, 300, 350, 500, or 1000 contiguous amino acids fromthe sequence shown in FIG. 2 (SEQ ID NO:2). Some fragments contain fewerthan 1000, 500, 250, 100 or 50 contiguous amino acids from SEQ ID NO:2.For example, exemplary fragments include fragments having 15-50 aminoacids or 100-500 amino acids. Some fragments include a motor domain oran active portion thereof. The motor domain runs from about amino acid402 to 727 of SEQ ID NO:2. Some fragments include a ligand bindingdomain of HsKifC2. Nucleic acids encoding such fragments are alsoincluded in the invention.

[0077] As further described herein, a wide variety of assays,therapeutic and diagnostic methods are provided herein which utilize thenovel compounds described herein. The uses and methods provided herein,as further described below have in vivo, in situ, and in vitroapplications, and can be used in medicinal, veterinary, agricultural andresearch based applications.

[0078] III. Isolation of the Gene Encoding HsKifC2

[0079] A. General Recombinant DNA Methods

[0080] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)), Methods in EnzymologyVol 266 (R. F. Doolittle, ed., 1996).

[0081] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from mass spectroscopy, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

[0082] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange BPLC as described in Pearson &Reanier, J. Chrom. 225:137-149 (1983).

[0083] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene16:21-26 (1981).

[0084] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding HsKifC2

[0085] In general, the nucleic acid sequences encoding HsKifC2 andrelated nucleic acid sequence homologs are cloned from cDNA and genomicDNA libraries or isolated using amplification techniques witholigonucleotide primers. Alternatively, expression libraries can be usedto clone HsKifC2 and HsKifC2 homologues by detected expressed homologuesimmunologically with antisera or purified antibodies made againstHsKifC2 that also recognize and selectively bind to the HsKifC2homologue. Finally, amplification techniques using primers can be usedto amplify and isolate HsKifC2 from DNA or RNA. Amplification techniquesusing degenerate primers can also be used to amplify and isolate HsKifC2homologues. Amplification techniques using primers can also be used toisolate a nucleic acid encoding HsKifC2. These primers can be used,e.g., to amplify a probe of several hundred nucleotides, which is thenused to screen a library for full-length HsKifC2.

[0086] Appropriate primers and probes for identifying the gene encodinghomologues of HsKifC2 in other species are generated from comparisons ofthe sequences provided herein. As described above, antibodies can beused to identify HsKifC2 homologues. For example, antibodies made to themotor domain of HsKifC2 or to the whole protein are useful foridentifying HsKifC2 homologues.

[0087] To make a cDNA library, one should choose a source that is richin the mRNA of choice, e.g., HsKifC2. For example, HsKifC2 tissuedistribution is dominant in colon, skin, and heart. See, FIG. 5. ThemRNA is then made into cDNA using reverse transcriptase, ligated into arecombinant vector, and introduced into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269); Sambrook et al., supra; Ausubel et al., supra).

[0088] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization is readout as generally described in Grunstein et al., Proc. Natl. Acad. Sci.USA, 72:3961-3965 (1975).

[0089] An alternative method of isolating HsKifC2 nucleic acid and itshomologues combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A guide to Methods and Applications (Inniset al., eds. 1990)). Methods such as polymerase chain reaction andligase chain reaction can be used to amplify nucleic acid sequences ofHsKifC2 directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplifyHsKifC2 homologues using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of HsKifC2 encoding mRNA in physiologicalsamples, for nucleic sequencing or for other purposes. Genes amplifiedby the PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

[0090] Gene expression of HsKifC2 can also be analyzed by techniquesknown in the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A+RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, quantitative PCR, and the like.

[0091] Synthetic oligonucleotides can be used to construct recombinantHsKifC2 genes for use as probes or for expression of protein. Thismethod is performed using a series of overlapping oligonucleotidesusually 40-120 bp in length, representing both the sense and nonsensestrands of the gene. These DNA fragments are then annealed, ligated andcloned. Alternatively, amplification techniques can be used with preciseprimers to amplify a specific subsequence of the HsKifC2 gene. Thespecific subsequence is then ligated into an expression vector.

[0092] The gene for HsKifC2 is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. The intermediate vectors are typicallyprokaryote vectors or shuttle vectors.

[0093] C. Expression in Prokaryotes and Eukaryotes

[0094] To obtain high level expression of a cloned gene, such as thosecDNAs encoding HsKifC2, it is important to construct an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing the HsKifC2 protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The pET expression system (Novagen) isa preferred prokaryotic expression system.

[0095] The promoter used to direct expression of a heterologous nucleicacid depends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

[0096] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the HsKifC2encoding nucleic acid in host cells. A typical expression cassette thuscontains a promoter operably linked to the nucleic acid sequenceencoding HsKifC2 and signals required for efficient polyadenylation ofthe transcript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding HsKifC2 may typically be linked to acleavable signal peptide sequence to promote secretion of the encodedprotein by the transformed cell. Such signal peptides would include,among others, the signal peptides from tissue plasminogen activator,insulin, and neuron growth factor, and juvenile hormone esterase ofHeliothis virescens. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

[0097] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0098] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc or histidine tags.

[0099] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, cytomegalovirus vectors, papilloma virus vectors, and vectorsderived from Epstein Bar virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

[0100] Some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. Alternatively, high yield expressionsystems not involving gene amplification are also suitable, such asusing a baculovirus vector in insect cells, with a HsKifC2 encodingsequence under the direction of the polyhedrin promoter or other strongbaculovirus promoters.

[0101] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical, any of themany resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0102] Standard transfection or transformation methods are used toproduce bacterial, mammalian, yeast or insect cell lines that expresslarge quantities of HsKifC2 protein, which are then purified usingstandard techniques (see, e.g., Colley et al., J. Biol. Chem.264:17619-17622 (1989); Guide to Protein Purification, in Methods inEnzymology, vol. 182 (Deutscher ed., 1990)).

[0103] Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds., 1983). Any of the well known proceduresfor introducing foreign nucleotide sequences into host cells may beused. These include the use of calcium phosphate transfection,polybrene, protoplast fusion, electroporation, liposomes,microinjection, plasma vectors, viral vectors and any of the other wellknown methods for introducing cloned genomic DNA, cDNA, synthetic DNA orother foreign genetic material into a host cell (see, e.g., Sambrook etal., supra). It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one gene into the host cell capable of expressing HsKifC2.

[0104] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofHsKifC2, which is recovered from the culture using standard techniquesidentified below.

[0105] IV. Purification of HsKifC2 Protein

[0106] Either naturally occurring or recombinant HsKifC2 can be purifiedfor use in functional assays. In a preferred embodiment, the targetproteins are purified for use in the assays to provide substantiallypure samples. Alternatively, the target protein need not besubstantially pure as long as the sample comprising the target proteinis substantially free of other components that can contribute to theproduction of ADP or phosphate.

[0107] The target proteins may be isolated or purified in a variety ofways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, chromatofocussing, selectiveprecipitation with such substances as ammonium sulfate; and others (see,e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S.Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).For example, the target protein can be purified using a standardanti-target antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.A preferred method of purification is use of Ni-NTA agarose (Qiagen).

[0108] The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubule gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1985)), as described in detailbelow.

[0109] A. Purification of HsKifC2 from Recombinant Bacteria

[0110] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a preferred method ofexpression. Bacteria are grown according to standard procedures in theart. Fresh or frozen bacteria cells are used for isolation of protein.

[0111] Alternatively, it is possible to purify HsKifC2 from bacteriaperiplasm. After HsKifC2 is exported into the periplasm of the bacteria,the periplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0112] Suitable purification schemes for some specific kinesins areoutlined in U.S. Ser. No. 09/295,612, filed Apr. 20, 1999, herebyexpressly incorporated herein in its entirety for all purposes.

[0113] B. Standard Protein Separation Techniques For Purifying HsKifC2

[0114] Solubility Fractionation

[0115] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0116] Size Differential Filtration

[0117] The molecular weight of HsKifC2 can be used to isolated it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

[0118] Column Chromatography

[0119] HsKifC2 can also be separated from other proteins on the basis ofits size, net surface charge, hydrophobicity, and affinity for ligands.In addition, antibodies raised against proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

[0120] V. Immunological Detection of HsKifC2

[0121] In addition to the detection of HsKifC2 genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect HsKifC2. Immunoassays can be used toqualitatively or quantitatively analyze HsKifC2. A general overview ofthe applicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

[0122] A. Antibodies to HsKifC2

[0123] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with HsKifC2 are known to those of skill in the art(see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow &Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986); and Kohler & Milstein, Nature 256:495497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

[0124] Humanized forms of mouse antibodies can be generated by linkingthe CDR regions of non-human antibodies to human constant regions byrecombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA86, 10029-10033 (1989) and WO 90/07861 (incorporated by reference forall purposes). Human antibodies can be obtained using phage-displaymethods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO92/01047. In these methods, libraries of phage are produced in whichmembers display different antibodies on their outersurfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to HsKifC2 or fragments thereof. Human antibodies againstHsKifC2 can also be produced from non-human transgenic mammals havingtransgenes encoding at least a segment of the human immunoglobulin locusand an inactivated endogenous immunoglobulin locus. See, e.g., Lonberget al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each ofwhich is incorporated by reference in its entirety for all purposes).Human antibodies can be selected by competitive binding experiments, orotherwise, to have the same epitope specificity as a particular mouseantibody. Such antibodies are particularly likely to share the usefulfunctional properties of the mouse antibodies. Human polyclonalantibodies can also be provided in the form of serum from humanimmunized with an immunogenic agent. Optionally, such polyclonalantibodies can be concentrated by affinity purification using HsKifC2 asan affinity reagent.

[0125] A number of HsKifC2 comprising immunogens may be used to produceantibodies specifically reactive with HsKifC2. For example, recombinantHsKifC2 or a antigenic fragment thereof such as the motor domain, isisolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.

[0126] Alternatively, a synthetic peptide derived from the sequencesdisclosed herein and conjugated to a carrier protein can be used animmunogen. Naturally occurring protein may also be used either in pureor impure form. The product is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated, for subsequent use in immunoassays to measure the protein.

[0127] Methods of production of polyclonal antibodies are known to thoseof skill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to HsKifC2. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

[0128] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989).

[0129] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against non-HsKifC2proteins or even other homologous proteins from other organisms (e.g.,C. elegans unc-104 or human Kif1A), using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

[0130] Once HsKifC2 specific antibodies are available, HsKifC2 can bedetected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio ed., 1980); and Harlow & Lane, supra.

[0131] B. Binding Assays

[0132] Antibodies can be used for treatment or to identify the presenceof HsKifC2 having the sequence identity characteristics as describedherein. Additionally, antibodies can be used to identify modulators ofthe interaction between the antibody and HsKifC2 as further describedbelow. While the following discussion is directed toward the use ofantibodies in the use of binding assays, it is understood that the samegeneral assay formats such as those described for “non-competitive” or“competitive” assays can be used with any compound which binds toHsKifC2 such as microtubules or the compounds described in Serial No.60/070,772.

[0133] In a preferred embodiment, HsKifC2 is detected and/or quantifiedusing any of a number of well recognized immunological binding assays(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a review of the general immunoassays, see also Methodsin Cell Biology Volume 37: Antibodies in Cell Biology (Asai, ed. 1993);Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe HsKifC2 or antigenic subsequence thereof). The antibody (e.g.,anti-HsKifC2) may be produced by any of a number of means well known tothose of skill in the art and as described above.

[0134] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeledHsKifC2 polypeptide or a labeled anti-HsKifC2 antibody. Alternatively,the labeling agent may be a third moiety, such a secondary antibody,that specifically binds to the antibody/HsKifC2 complex (a secondaryantibody is typically specific to antibodies of the species from whichthe first antibody is derived). Other proteins capable of specificallybinding immunoglobulin constant regions, such as protein A or protein Gmay also be used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see generally Kronval et al., J. Immunol.111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135:2589-2542(1985)). The labeling agent can be modified with a detectable moiety,such as biotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

[0135] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 4° C. to 40°C.

[0136] Non-Competitive Assay Formats

[0137] Immunoassays for detecting HsKifC2 in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-HsKifC2 antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture HsKifC2 present in the test sample.HsKifC2 is thus immobilized is then bound by a labeling agent, such as asecond HsKifC2 antibody bearing a label. Alternatively, the secondantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. The second or third antibody is typicallymodified with a detectable moiety, such as biotin, to which anothermolecule specifically binds, e.g., streptavidin, to provide a detectablemoiety.

[0138] Competitive Assay Formats

[0139] In competitive assays, the amount of HsKifC2 present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) HsKifC2 displaced (competed away) from an anti-HsKifC2antibody by the unknown HsKifC2 present in a sample. In one competitiveassay, a known amount of HsKifC2 is added to a sample and the sample isthen contacted with an antibody that specifically binds to HsKifC2. Theamount of exogenous HsKifC2 bound to the antibody is inverselyproportional to the concentration of HsKifC2 present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of HsKifC2 bound to the antibody may bedetermined either by measuring the amount of HsKifC2 present in aHsKifC2/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of HsKifC2 may be detected byproviding a labeled HsKifC2 molecule.

[0140] A hapten inhibition assay is another preferred competitive assay.In this assay the known HsKifC2, is immobilized on a solid substrate. Aknown amount of anti-HsKifC2 antibody is added to the sample, and thesample is then contacted with the HsKifC2. The amount of anti-HsKifC2antibody bound to the known immobilized HsKifC2 is inverselyproportional to the amount of HsKifC2 present in the sample. Again, theamount of immobilized antibody may be detected by detecting either theimmobilized fraction of antibody or the fraction of the antibody thatremains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

[0141] Cross-reactivity Determinations

[0142] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2 can be immobilized to a solid support.Proteins (e.g., C. elegans unc-104 or human Kif1A) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability ofHsKifC2 encoded by SEQ ID NO:2 to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologues.

[0143] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps the protein of this invention, to theimmunogen protein (i.e., HsKifC2 of SEQ ID NO:2). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to a HsKifC2immunogen.

[0144] Other Assay Formats

[0145] Western blot (immunoblot) analysis is used to detect and quantifythe presence of HsKifC2 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind HsKifC2. The anti-HsKifC2 antibodies specificallybind to the HsKifC2 on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-HsKifC2 antibodies.

[0146] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

[0147] Reduction of Non-specific Binding

[0148] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0149] Labels

[0150] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), calorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.) or other labels that can be detected by massspectroscopy, NMR spectroscopy, or other analytical means known in theart.

[0151] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0152] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize HsKifC2, or secondary antibodies that recognizeanti-HsKifC2.

[0153] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

[0154] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0155] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0156] VI. Assays for Modulators of the Target Protein

[0157] A. Functional Assays

[0158] The activity of biologically active HsKifC2 can be assessed usinga variety of in vitro or in vivo assays known in the art, e.g., ATPase,microtubule gliding, and microtubule binding, and microtubuledepolymerization assays (Kodama et al., J. Biochem. 99: 1465-1472(1986); Stewart et al, Proc. Nat'l Acad. Sci. USA 90: 5209-5213 (1993);(Lombillo et al., J. Cell Biol. 128:107-115 (1995); (Vale et al., Cell42:39-50 (1985)). Methods of performing motility assays are well known(see, e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476, Turner etal., 1996, Anal. Biochem. 242 (1):20-5; Gittes et al., 1996, Biophys. J.70(1): 418-29; Shirakawa et al., 1995, J. Exp. Biol. 198: 1809-15;Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann et al.,1995, Biophys. J. 68: 72S, and the like).

[0159] A preferred assay for high throughput screening is an ATPaseassay with colorimetric detection, e.g., malachite green for end-pointdetection or coupled PK/LDH for continuous rate monitoring. An exemplaryATPase activity assay utilizes 0.3 M PCA (perchloric acid) and malachitegreen reagent (8.27 mM sodium molybdate II, 0.33 mM malachite greenoxalate, and 0.8 mM Triton X-100). To perform the assay, 10 μL ofreaction is quenched in 90 μL of cold 0.3 M PCA. Phosphate standards areused so data can be converted to mM inorganic phosphate released. Whenall reactions and standards have been quenched in PCA, 100 μL ofmalachite green reagent is added to the to relevant wells in e.g., amicrotiter plate. The mixture is developed for 10-15 minutes and theplate is read at an absorbance of 650 nm. If phosphate standards wereused, absorbance readings can be converted to mM Pi and plotted overtime. Additionally, ATPase assays known in the art include theluciferase assay.

[0160] Another exemplary assay can be performed using the following twospecific solutions. Solution A contains 1 mM ATP, 2 mMphosphoenolpyruvate in a working buffer (25 mM Pipes pH 6.8, 2 mM MgCl2,1 mM EGTA, 1 mM DTT, 5 μM taxol, 25 ppm Antifoam, pH 6.8. Solution Bcontains 0.6 mM NADH, 0.2 mg/ml BSA, 1:100 dilution of PK/LDH mixturefrom Sigma, 200 μg/ml microtubules, 100 nM HsKifC2. To initiate theexperiment, 1 μl of DMSO stock of test compounds is added to each wellof the bottom row of a 96-well half area plate. Control wells containonly DMSO alone. 50 μl of solution A is then added to each well. Thesolutions are mixed by repeated pipetting, followed by a series ofdilution by repeated transferring of 50 μl of solution between rows. Thereaction is initiated by adding 50 μl of solution B. The plate is theninserted in the reader and absorbance at 340 nM was monitored for 5 min.The observed rate for 50 μl Solution A+50 μl Solution B in a half-areaplate should be about 100 mOD/min. Optionally, a series of dilution ismade and absorbance similarly measured. Similar procedures can be usedto study the inhibitory effect of a test agent on the basal (i.e., notmicrotubule-dependent) ATPase of HsKifC2. In these assays, microtubulesare omitted from Solution B, and HsKifC2 concentration is increased toat least 2 mM.

[0161] Such assays can be used to test for the activity of HsKifC2isolated from endogenous sources or recombinant sources. Furthermore,such assays can be used to test for modulators of HsKifC2. Modulatorscan increase or decrease activity of HsKifC2. Modulation is tested byscreening for candidate agents capable of modulating the activity of thetarget protein comprising the steps of combining a candidate agent withthe target protein, as above, and determining an alteration in thebiological activity of the target protein. Thus, in this embodiment, thecandidate agent should both bind to the target protein (although thismay not be necessary), and alter its biological or biochemical activityas defined herein. The methods include both in vitro screening methodsand in vivo screening of cells for alterations in cell cycledistribution, cell viability, or for the presence, morphology, activity,distribution, or amount of mitotic spindles, as are generally outlinedabove.

[0162] In a preferred embodiment, molecular motor activity is measuredby the methods disclosed in Ser. No. 09/314,464, filed May 18, 1999,entitled “Compositions and assay utilizing ADP or phosphate fordetecting protein modulators”, which is incorporated herein by referencein its entirety. More specifically, this assay detects modulators of anyaspect of a kinesin motor function ranging from interaction withmicrotubules to hydrolysis of ATP. ADP or phosphate is used as thereadout for protein activity.

[0163] There are a number of enzymatic assays known in the art which useADP as a substrate. For example, kinase reactions such as pyruvatelinases are known. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31(1970). This is a preferred method in that it allows the regeneration ofATP. In one embodiment, the level of activity of the enzymatic reactionis determined directly. In a preferred embodiment, the level of activityof the enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction. For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art. Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

[0164] In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

[0165] In addition, target protein activity can be examined bydetermining modulation of target protein in vitro using cultured cells.The cells are treated with a candidate agent and the effect of suchagent on the cells is then determined either directly or by examiningrelevant surrogate markers. For example, characteristics such as mitoticspindle morphology and cell cycle distribution can be used to determinethe effect.

[0166] Thus, in a preferred embodiment, the methods comprise combining atarget protein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

[0167] As will be appreciated by those in the art, the components may beadded in buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

[0168] In a preferred embodiment, an antifoam or a surfactant isincluded in the assay mixture. Suitable antifoams include, but are notlimited to, antifoam 289 (Sigma). Suitable surfactants include, but arenot limited to, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

[0169] A preferred assay design is also provided. In one aspect, theinvention provides a multi-time-point (kinetic) assay, with at least twodata points being preferred. In the case of multiple measurements, theabsolute rate of the protein activity can be determined.

[0170] B. Binding Assays

[0171] In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

[0172] Competitive screening assays may be done by combining the targetprotein and a drug candidate in a first sample. A second samplecomprises a candidate agent, the target protein and a compound that isknown to modulate the target protein. This may be performed in eitherthe presence or absence of microtubules. The binding of the candidateagent is determined for both samples, and a change, or difference inbinding between the two samples indicates the presence of an agentcapable of binding to the target protein and potentially modulating itsactivity. That is, if the binding of the candidate agent is different inthe second sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

[0173] In one embodiment, the candidate agent is labeled. Either thecandidate agent, or the competitor, or both, is added first to thetarget protein for a time sufficient to allow binding. Incubations maybe performed at any temperature which facilitates optimal activity,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapid highthroughput screening. Typically between 0.1 and 1 hour will besufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

[0174] In a preferred embodiment, the competitor is added first,followed by the candidate agent. Displacement of the competitor is anindication the candidate agent is binding to the target protein and thusis capable of binding to, and potentially modulating, the activity ofthe target protein. In this embodiment, either component can be labeled.Thus, for example, if the competitor is labeled, the presence of labelin the wash solution indicates displacement by the agent. Alternatively,if the candidate agent is labeled, the presence of the label on thesupport indicates displacement.

[0175] In an alternative embodiment, the candidate agent is added first,with incubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

[0176] C. Candidate Agents

[0177] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

[0178] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. In a preferredembodiment, the candidate agents are organic chemical moieties, a widevariety of which are available in the literature. Combinatoriallibraries can be produced for many types of compounds that can besynthesized in a step-by-step fashion. Such compounds includepolypeptides, proteins, nucleic acids, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines and oligocarbamates. Large combinatoriallibraries of compounds can be constructed by the encoded syntheticlibraries (ESL) method described in Affymax, WO 95/12608, Affymax WO93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503and Scripps, WO 95/30642 (each of which is incorporated herein byreference in its entirety for all purposes). Peptide libraries can alsobe generated by phage display methods. See, e.g., Devlin, WO 91/18980.Compounds to be screened can also be obtained from governmental orprivate sources, including, e.g., the National Cancer Institute's (NCI)Natural Product Repository, Bethesda, Md., the NCI Open SyntheticCompound Collection, Bethesda, Md., NCI's Developmental TherapeuticsProgram, or the like. Compounds to be screened can also be obtained fromgovernmental or private sources, including, e.g., the National CancerInstitute's (NCI) Natural Product Repository, Bethesda, Md., the NCIOpen Synthetic Compound Collection, Bethesda, Md., NCI's DevelopmentalTherapeutics Program, or the like.

[0179] D. Other Assay Components

[0180] The assays provided utilize target protein as defined herein. Inone embodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

[0181] A variety of other reagents may be included in the screeningassays. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Also, reagents that otherwise improve the efficiency ofthe assay, such as protease inhibitors, nuclease inhibitors,anti-microbial agents, etc., may be used. The mixture of components maybe added in any order that provides for the requisite binding.

[0182] VII. Applications

[0183] The methods of the invention are used to identify compoundsuseful in the treatment of cellular proliferation diseases. Diseasestates which can be treated by the methods and compositions providedherein include, but are not limited to, cancer (further discussedbelow), autoimmune disease, arthritis, graft rejection, inflammatorybowel disease, proliferation induced after medical procedures,including, but not limited to, surgery, angioplasty, and the like. It isappreciated that in some cases the cells may not be in a hyper or hypoproliferation state (abnormal state) and still require treatment. Forexample, during wound healing, the cells may be proliferating“normally”, but proliferation enhancement may be desired. Similarly, asdiscussed above, in the agriculture arena, cells may be in a “normal”state, but proliferation modulation may be desired to enhance a crop bydirectly enhancing growth of a crop, or by inhibiting the growth of aplant or organism which adversely affects the crop. Thus, in oneembodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

[0184] The compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc.More particularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cel, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinorna,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteocbronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinaoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic:blood (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;and Adrenal glands: neuroblastoma Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of the aboveidentified conditions.

[0185] Accordingly, the compositions of the invention are administeredto cells. By “administered” herein is meant administration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

[0186] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals, and otherorganisms. Thus the methods are applicable to both human therapy andveterinary applications. In the preferred embodiment the patient is amammal, and in the most preferred embodiment the patient is human.

[0187] Candidate agents having the desired pharmacological activity maybe administered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agent maybe administered alone or incombination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

[0188] In a preferred embodiment, the pharmaceutical compositions are ina water soluble form, such as pharmaceutically acceptable salts, whichis meant to include both acid and base addition salts.

[0189] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

[0190] The administration of the candidate agents of the presentinvention can be done in a variety of ways as discussed above,including, but not limited to, orally, subcutaneously, intravenously,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. In someinstances, for example, in the treatment of wounds and inflammation, thecandidate agents may be directly applied as a solution or spray.

[0191] One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

[0192] The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

[0193] HsKifC2's microtubule motor activity makes it useful for avariety of other applications. The kinesins of the present invention andin particular their motor domains can be used for separation of aspecific ligand from a heterologous mixtures in aqueous solution asdescribed by Stewart (U.S. Pat. No. 5,830,659). In the system discussedby Stewart, a kinesin motor domain is linked to a ligand binding moiety,such as streptavidin. The chimeric kinesin motor domains are placed intoa loading chamber containing the heterogeneous mixtures which is coupledto a receiving chamber by a channel bearing immobilized, alignedmicrotubules. Addition of ATP to the loading chamber results intranslocation of the kinesin motor domains, now attached non-covalentlyto the desired ligand via their ligand binding moiety, from the loadingchamber to the receiving chamber. Hence, the ATP-driven motilityactivity of the kinesin motor domain results in separation of thedesired ligand from the heterogeneous mixture. According to Stewart, allkinesin motor domains are suitable for use in the separation system.

[0194] The kinesins of the present invention and in particular the motordomains can also be used in nanotechnological applications. In general,the ability to convert chemical energy to linear force exerted upon amicrotubule substrate is useful in the design of nanoscale switchingdevices and nanometer scale rotory devices (see Komgruth, Nanotechnologyand Biomolecular Electronics, National Institute of Standards andTechnology White Paper). Kinesin motor domains (e.g., KifC2 motor domaindescribed above) may be used in the construction of rotors and othermechanical components (for review see Limberis and Stewart,Nanotechnology 11:47-51 (2000)); by linking ATP synthesis to aphotosynthetic process, kinesin motor domains may be employed aslight-operated molecular shuttles useful for nanoscale switches andpumps (see Dennis et al., Nanotechnology 10:232-236 (1999)). Additionalapplications include cascaded biomolecular linearmotoric motilitysystems, wherein a microtubule stator and kinesin slide are utilized inthe design of an actuator (see Stracke et al., Nanotechnology 11:52-56(2000)) and linear analysis of polymers (see U.S. Pat. No. 6,210,896).

[0195] Nucleic acids encoding the KifC2 protein or fragments thereof arealso useful for inclusion on a GeneChip™ array or the like for use inexpression monitoring (see U.S. Pat. No. 6,040,138, EP 853, 679 andWO97/27317). Such arrays typically contain oligonucleotide or cDNAprobes to allow detection of large numbers of mRNAs within a mixture.Many of the nucleic acids included in such arrays are from genes or ESTsthat have not been well characterized. Such arrays are often used tocompare expression profiles between different tissues or betweendifferent conditions of the same tissue (healthy vs. diseased ordrug-treated vs. control) to identify differentially expressedtranscripts. The differentially expressed transcripts are then usefule.g., for diagnosis of disease states, or to characterize responses ofdrugs. The nucleic acids of the invention can be included on GeneChip™arrays or the like together with probes containing a variety of othergenes. The present nucleic acids are particularly useful for inclusionin GeneChip™ arrays for analyzing the cell cycle or proliferation stateof cells. Nucleic acids encoding HsKifC2 can be combined with nucleicacids encoding other kinesin molecules and/or nucleic acids from othergenes having roles in DNA replication, cell division or other cell cyclefunction. Such arrays are useful for analyzing and diagnosing cells in aproliferating state, and diseases such as cancer characterized bypresence of the same. Such arrays are also useful for analyzingcandidate drugs for roles in modulation of the cell cycle andproliferation. The efficacy of such drugs can be assayed by determiningthe effect of the drug on the expression profile of genes affectingproliferation and the cell cycle.

[0196] VIII. Examples

[0197] Assay for Detecting and Monitoring HsKifC2 ATPase Activity

[0198] This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATP production is monitored bya coupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekinase catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and AND⁺.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 nm.

[0199] The final 25 μl assay solution consists of the following: 5 μg/mltarget protein, 30 μg/ml microtubules, 5 μM Taxol 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2 mM MgCl₂, 1 mM EGTA, 1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

[0200] Potential candidate agents are dissolved in DMSO at aconcentration of about 1 mg/ml and 0.5 μl of each chemical solution isdispensed into a single well of a clear 384 well plate. Each of the 384wells are then filled with 20 μl of a solution consisting of all of theassay components described above except for ATP. The plate is agitatedat a high frequency. To start the assay, 5 μl of a solution containingATP is added to each well. The plate is agitated and the absorbance isread at 340 nm over various time intervals. The assay is run at roomtemperature.

[0201] The assay components and the performance of the assay areoptimized together to match the overall read time with the rate of thetarget protein's ADP production. The read time should be long enough forthe rate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

[0202] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety.

1 4 1 2373 DNA Homo sapiens human kinesin motor protein HsKifC2 1cccatgtacg ccttttactc gttgctcatc tacatcttct acagcctctt ccgcagggat 60ggtggcgccg cggcggccgc ggagcccggg gaccccgccc agccccgggg tcgccggcgc 120ccagacctgc ccgcgccaga gctgtggacc gagctgaccg gcctggccgg tagctccgag 180cctgaggatg ggtcggaagg cgcagccgag ggtcgcgcgg ccgcggtgtc cctggaagag 240gccctactgc gcctcgccga gttcctctcc gtccagctgg gggcggaaga gagctgcggg 300ggcccggcgg acctgggcca gtctggcgag gtcccctcac tgttgacagt gaccagtcag 360ctcttggccc ttctggcatg gcttcgaagc cccaggggga ggcaggccct gctccagggg 420actcagccag cccctcgggt ccggcccccc tctccagatg gatccacatc ccaagaagaa 480agcccttccc acttcaccgc agtcccaggc gagccactgg gggatgagac ccagggacag 540cagcccctcc agttggagga ggatcagagg gcgtggcagc ggctggagca gctcatcctg 600ggacagctgg aggagctgaa gcagcagctg gaacagcagg aggaggagtt gggtcgactg 660cgcctgggcg tgggggcgac ggactcagag aaaagggttc agcatctgac tctggagaac 720gaggccctga agcagagcct gagtctcatg cgggacctcc tgctgcactg gggccccggg 780ccccccatca gggctccgca ggaggaggcg gaggcattgc tagagctcca gggccggctt 840caggaggccc aagacaccac agaagccctc cgagcccagc tgggggtgca ggaggtgcag 900ctgcagggcc ttcaaggggc cctccagcag ctccagcagg agacggagca gaactgcagg 960cgtgagctac agcagatgca tgggcagctg gcaggacttc gggcacggat ggccagcctg 1020cgtcagggct gcggggacct ccgaggtttg gtcagcacct ttacccagag ctgtcagggt 1080tcgctgagtg aggcccgggg ccaggtgtcc tgggccttgg gggcactgtc atctggaggg 1140cctggcactc agctccctga ggggcagcaa gggcccccag ccggatgccc agggcggctg 1200ccagaactca agggaaatat ccgtgtgctg tgtcggctga ggccagggac atcttctagc 1260cttgtgagtg tggagcctgg cccagggggc accgtcacca cctgctaccg ggggcgccat 1320cgtcgattcc gcctagactg ggtcttccct ccagacgcca gccaggagga ggtcttcaga 1380gagctggaac ctgcggtgct gtcctgcctc cgaggctaca gcgtctgcat cttcacctat 1440ggccagacag gcaccgggaa gacctacagc atggagggcc ctcctgagga ccccggcata 1500gttcctaggg cgctgcagtc gctgttccgg gagatggggg ccggccggca gcaccgggtg 1560acactcagca tggtggagat ctacaatgag gctgtcaggg acctccttgc tccagggcct 1620cccgagcgcc tggccgtgag gcagggccca gaaggccagg gcgggatcca ggtggctggc 1680ctcacccact gggacgtgcc caacctggag acattgcacc agatgctgaa actggggagg 1740agcaaccggg ccaccgccgc caccgccatg aaccagcgca gctcccgctc gcatgccctg 1800gtcacgctga cgctgcgcgc ggcgtctcca ccgcgcgctc caggcaccgc aggcacgctg 1860cacctggtgg acctggcggg atccgaacgc gcacggaagg caggggcggt cggcccgccg 1920cggggagacc cagacggcgc ccggcgcctg cgggaggccc agaccataaa ccgctcgctg 1980ctggcgctag gaggcgtgat ggccgcactg cgggcccacc ggccgcacgt gcccttccgc 2040gactcgcagc tcacgcgact gctgcagccg gcgctgggcc caggcaccac cgcggtgctg 2100ctgctgcaga tctccacgcg gccggaggat ctcggggaga cagtctgctc cctcaagttc 2160gccgaccgag tgggtcaagt ggagctgggg ccagcccggc gccgcagggt cccgcgctcc 2220tccgggacgc cttcttccct cagcaccgac actccgctca ccgggacccc ctgcacccct 2280acgccgtccc ctggcagtcc tccatgcccc agtcccgaca acggctcggg ctcggctctc 2340gcgcccgcag agggcctgcc cctctagtcc tgg 2373 2 787 PRT Homo sapiens humankinesin motor protein HsKifC2 2 Met Tyr Ala Phe Tyr Ser Leu Leu Ile TyrIle Phe Tyr Ser Leu Phe 1 5 10 15 Arg Arg Asp Gly Gly Ala Ala Ala AlaAla Glu Pro Gly Asp Pro Ala 20 25 30 Gln Pro Arg Gly Arg Arg Arg Pro AspLeu Pro Ala Pro Glu Leu Trp 35 40 45 Thr Glu Leu Thr Gly Leu Ala Gly SerSer Glu Pro Glu Asp Gly Ser 50 55 60 Glu Gly Ala Ala Glu Gly Arg Ala AlaAla Val Ser Leu Glu Glu Ala 65 70 75 80 Leu Leu Arg Leu Ala Glu Phe LeuSer Val Gln Leu Gly Ala Glu Glu 85 90 95 Ser Cys Gly Gly Pro Ala Asp LeuGly Gln Ser Gly Glu Val Pro Ser 100 105 110 Leu Leu Thr Val Thr Ser GlnLeu Leu Ala Leu Leu Ala Trp Leu Arg 115 120 125 Ser Pro Arg Gly Arg GlnAla Leu Leu Gln Gly Thr Gln Pro Ala Pro 130 135 140 Arg Val Arg Pro ProSer Pro Asp Gly Ser Thr Ser Gln Glu Glu Ser 145 150 155 160 Pro Ser HisPhe Thr Ala Val Pro Gly Glu Pro Leu Gly Asp Glu Thr 165 170 175 Gln GlyGln Gln Pro Leu Gln Leu Glu Glu Asp Gln Arg Ala Trp Gln 180 185 190 ArgLeu Glu Gln Leu Ile Leu Gly Gln Leu Glu Glu Leu Lys Gln Gln 195 200 205Leu Glu Gln Gln Glu Glu Glu Leu Gly Arg Leu Arg Leu Gly Val Gly 210 215220 Ala Thr Asp Ser Glu Lys Arg Val Gln His Leu Thr Leu Glu Asn Glu 225230 235 240 Ala Leu Lys Gln Ser Leu Ser Leu Met Arg Asp Leu Leu Leu HisTrp 245 250 255 Gly Pro Gly Pro Pro Ile Arg Ala Pro Gln Glu Glu Ala GluAla Leu 260 265 270 Leu Glu Leu Gln Gly Arg Leu Gln Glu Ala Gln Asp ThrThr Glu Ala 275 280 285 Leu Arg Ala Gln Leu Gly Val Gln Glu Val Gln LeuGln Gly Leu Gln 290 295 300 Gly Ala Leu Gln Gln Leu Gln Gln Glu Thr GluGln Asn Cys Arg Arg 305 310 315 320 Glu Leu Gln Gln Met His Gly Gln LeuAla Gly Leu Arg Ala Arg Met 325 330 335 Ala Ser Leu Arg Gln Gly Cys GlyAsp Leu Arg Gly Leu Val Ser Thr 340 345 350 Phe Thr Gln Ser Cys Gln GlySer Leu Ser Glu Ala Arg Gly Gln Val 355 360 365 Ser Trp Ala Leu Gly AlaLeu Ser Ser Gly Gly Pro Gly Thr Gln Leu 370 375 380 Pro Glu Gly Gln GlnGly Pro Pro Ala Gly Cys Pro Gly Arg Leu Pro 385 390 395 400 Glu Leu LysGly Asn Ile Arg Val Leu Cys Arg Leu Arg Pro Gly Thr 405 410 415 Ser SerSer Leu Val Ser Val Glu Pro Gly Pro Gly Gly Thr Val Thr 420 425 430 ThrCys Tyr Arg Gly Arg His Arg Arg Phe Arg Leu Asp Trp Val Phe 435 440 445Pro Pro Asp Ala Ser Gln Glu Glu Val Phe Arg Glu Leu Glu Pro Ala 450 455460 Val Leu Ser Cys Leu Arg Gly Tyr Ser Val Cys Ile Phe Thr Tyr Gly 465470 475 480 Gln Thr Gly Thr Gly Lys Thr Tyr Ser Met Glu Gly Pro Pro GluAsp 485 490 495 Pro Gly Ile Val Pro Arg Ala Leu Gln Ser Leu Phe Arg GluMet Gly 500 505 510 Ala Gly Arg Gln His Arg Val Thr Leu Ser Met Val GluIle Tyr Asn 515 520 525 Glu Ala Val Arg Asp Leu Leu Ala Pro Gly Pro ProGlu Arg Leu Ala 530 535 540 Val Arg Gln Gly Pro Glu Gly Gln Gly Gly IleGln Val Ala Gly Leu 545 550 555 560 Thr His Trp Asp Val Pro Asn Leu GluThr Leu His Gln Met Leu Lys 565 570 575 Leu Gly Arg Ser Asn Arg Ala ThrAla Ala Thr Ala Met Asn Gln Arg 580 585 590 Ser Ser Arg Ser His Ala LeuVal Thr Leu Thr Leu Arg Ala Ala Ser 595 600 605 Pro Pro Arg Ala Pro GlyThr Ala Gly Thr Leu His Leu Val Asp Leu 610 615 620 Ala Gly Ser Glu ArgAla Arg Lys Ala Gly Ala Val Gly Pro Pro Arg 625 630 635 640 Gly Asp ProAsp Gly Ala Arg Arg Leu Arg Glu Ala Gln Thr Ile Asn 645 650 655 Arg SerLeu Leu Ala Leu Gly Gly Val Met Ala Ala Leu Arg Ala His 660 665 670 ArgPro His Val Pro Phe Arg Asp Ser Gln Leu Thr Arg Leu Leu Gln 675 680 685Pro Ala Leu Gly Pro Gly Thr Thr Ala Val Leu Leu Leu Gln Ile Ser 690 695700 Thr Arg Pro Glu Asp Leu Gly Glu Thr Val Cys Ser Leu Lys Phe Ala 705710 715 720 Asp Arg Val Gly Gln Val Glu Leu Gly Pro Ala Arg Arg Arg ArgVal 725 730 735 Pro Arg Ser Ser Gly Thr Pro Ser Ser Leu Ser Thr Asp ThrPro Leu 740 745 750 Thr Gly Thr Pro Cys Thr Pro Thr Pro Ser Pro Gly SerPro Pro Cys 755 760 765 Pro Ser Pro Asp Asn Gly Ser Gly Ser Ala Leu AlaPro Ala Glu Gly 770 775 780 Leu Pro Leu 785 3 978 DNA Homo sapiens humankinesin motor protein HsKifC2 motor domain fragment 3 ctcaagggaaatatccgtgt gctgtgtcgg ctgaggccag ggacatcttc tagccttgtg 60 agtgtggagcctggcccagg gggcaccgtc accacctgct accgggggcg ccatcgtcga 120 ttccgcctagactgggtctt ccctccagac gccagccagg aggaggtctt cagagagctg 180 gaacctgcggtgctgtcctg cctccgaggc tacagcgtct gcatcttcac ctatggccag 240 acaggcaccgggaagaccta cagcatggag ggccctcctg aggaccccgg catagttcct 300 agggcgctgcagtcgctgtt ccgggagatg ggggccggcc ggcagcaccg ggtgacactc 360 agcatggtggagatctacaa tgaggctgtc agggacctcc ttgctccagg gcctcccgag 420 cgcctggccgtgaggcaggg cccagaaggc cagggcggga tccaggtggc tggcctcacc 480 cactgggacgtgcccaacct ggagacattg caccagatgc tgaaactggg gaggagcaac 540 cgggccaccgccgccaccgc catgaaccag cgcagctccc gctcgcatgc cctggtcacg 600 ctgacgctgcgcgcggcgtc tccaccgcgc gctccaggca ccgcaggcac gctgcacctg 660 gtggacctggcgggatccga acgcgcacgg aaggcagggg cggtcggccc gccgcgggga 720 gacccagacggcgcccggcg cctgcgggag gcccagacca taaaccgctc gctgctggcg 780 ctaggaggcgtgatggccgc actgcgggcc caccggccgc acgtgccctt ccgcgactcg 840 cagctcacgcgactgctgca gccggcgctg ggcccaggca ccaccgcggt gctgctgctg 900 cagatctccacgcggccgga ggatctcggg gagacagtct gctccctcaa gttcgccgac 960 cgagtgggtcaagtggag 978 4 326 PRT Homo sapiens human kinesin motor protein HsKifC2motor domain fragment 4 Leu Lys Gly Asn Ile Arg Val Leu Cys Arg Leu ArgPro Gly Thr Ser 1 5 10 15 Ser Ser Leu Val Ser Val Glu Pro Gly Pro GlyGly Thr Val Thr Thr 20 25 30 Cys Tyr Arg Gly Arg His Arg Arg Phe Arg LeuAsp Trp Val Phe Pro 35 40 45 Pro Asp Ala Ser Gln Glu Glu Val Phe Arg GluLeu Glu Pro Ala Val 50 55 60 Leu Ser Cys Leu Arg Gly Tyr Ser Val Cys IlePhe Thr Tyr Gly Gln 65 70 75 80 Thr Gly Thr Gly Lys Thr Tyr Ser Met GluGly Pro Pro Glu Asp Pro 85 90 95 Gly Ile Val Pro Arg Ala Leu Gln Ser LeuPhe Arg Glu Met Gly Ala 100 105 110 Gly Arg Gln His Arg Val Thr Leu SerMet Val Glu Ile Tyr Asn Glu 115 120 125 Ala Val Arg Asp Leu Leu Ala ProGly Pro Pro Glu Arg Leu Ala Val 130 135 140 Arg Gln Gly Pro Glu Gly GlnGly Gly Ile Gln Val Ala Gly Leu Thr 145 150 155 160 His Trp Asp Val ProAsn Leu Glu Thr Leu His Gln Met Leu Lys Leu 165 170 175 Gly Arg Ser AsnArg Ala Thr Ala Ala Thr Ala Met Asn Gln Arg Ser 180 185 190 Ser Arg SerHis Ala Leu Val Thr Leu Thr Leu Arg Ala Ala Ser Pro 195 200 205 Pro ArgAla Pro Gly Thr Ala Gly Thr Leu His Leu Val Asp Leu Ala 210 215 220 GlySer Glu Arg Ala Arg Lys Ala Gly Ala Val Gly Pro Pro Arg Gly 225 230 235240 Asp Pro Asp Gly Ala Arg Arg Leu Arg Glu Ala Gln Thr Ile Asn Arg 245250 255 Ser Leu Leu Ala Leu Gly Gly Val Met Ala Ala Leu Arg Ala His Arg260 265 270 Pro His Val Pro Phe Arg Asp Ser Gln Leu Thr Arg Leu Leu GlnPro 275 280 285 Ala Leu Gly Pro Gly Thr Thr Ala Val Leu Leu Leu Gln IleSer Thr 290 295 300 Arg Pro Glu Asp Leu Gly Glu Thr Val Cys Ser Leu LysPhe Ala Asp 305 310 315 320 Arg Val Gly Gln Val Glu 325

What is claimed is:
 1. An isolated nucleic acid sequence encoding amicrotubule motor protein, wherein the motor protein has the followingproperties: (i) the protein's activity includes microtubule stimulatedATPase activity; and (ii) the protein has a sequence that has greaterthan 70% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4 asmeasured using a sequence comparison algorithm.
 2. An isolated nucleicacid sequence of claim 1, wherein the encoded protein specifically bindsto polyclonal antibodies to a protein comprising SEQ ID NO:2 or SEQ IDNO:4.
 3. An isolated nucleic acid sequence of claim 1, wherein thenucleic acid encodes SEQ ID NO:2 or SEQ ID NO:4.
 4. An isolated nucleicacid sequence of claim 1, wherein the nucleic acid has a nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3.
 5. An isolated nucleic acidsequence of claim 1, wherein the nucleic acid selectively hybridizesunder stringent hybridization conditions to SEQ ID NO:1 or SEQ ID NO:3.6. An expression vector comprising a nucleic acid encoding a microtubulemotor protein, wherein the motor protein has the following properties:(i) the protein's activity includes microtubule stimulated ATPaseactivity; and (ii) the protein has a sequence that has greater than 70%amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measuredusing a sequence comparison algorithm.
 7. A host cell transfected withthe vector of claim
 6. 8. An isolated microtubule motor protein, whereinthe protein has greater than 70% amino acid sequence identity to SEQ IDNO:2 or SEQ ID NO:4 as measured using a sequence comparison algorithm.9. An isolated protein of claim 8, wherein the protein specificallybinds to polyclonal antibodies to HsKifC2.
 10. An isolated protein ofclaim 8, wherein the protein is HsKifC2.
 11. An isolated protein ofclaim 8, wherein the protein has an amino acid sequence of SEQ ID NO:2or SEQ ID NO:4.
 12. An isolated protein of claim 8, wherein the proteinspecifically binds to polyclonal antibodies generated against a motordomain of HsKifC2.
 13. An isolated protein of claim 8, wherein theprotein comprises an amino acid sequence of a HsKifC2 motor domain. 14.A method for screening for modulators of HsKifC2, the method comprisingthe steps of: (i) providing biologically active HsKifC2, wherein has thefollowing properties: (i) activity including microtubule stimulatedATPase activity; and (ii) sequence that has greater than 70% amino acidsequence identity to HsKifC2 of SEQ ID NO:2 or SEQ ID NO:4 as measuredusing a sequence comparison algorithm; (ii) contacting biologicallyactive HsKifC2 with a candidate agent in a test and controlconcentration; and (iii) assaying for the level of HsKifC2 activity,wherein the HsKifC2 activity is selected from the group consisting ofbinding activity or ATPase activity, and wherein a change in activitybetween the test and control concentration indicates a modulator.
 15. Amethod of claim 14, wherein the screening occurs in a multi-well plateas part of a high-throughput screen.
 16. A method of claim 14, whereinthe biologically active HsKifC2 comprises an amino acid sequence of aHsKifC2 motor domain.
 17. A compound that modulates HsKifC2, whereinsaid compound is identified using the method of claim
 14. 18. Anisolated nucleic acid comprising a sequence which has greater than 60%sequence identity with nucleotide SEQ ID NO:1 or SEQ ID NO:3.